Caffeine is the world's most popular psychoactive drug and a powerful modulator of excitability in the brain. Caffeine's stimulant properties depend on its ability to interact with neurotransmission and enhance neuronal activities by controlling a release of neurotransmitters. Yet despite its well-established stimulatory effect on excitatory neurotransmitter release, caffeine's effect on the release of the inhibitory neurotransmitter, GABA, is not completely understood. Findings from our laboratory suggest caffeine inhibits GABAergic transmission. Moreover, caffeine's effect can be amplified when cytosolic calcium ([Ca2+]i) is elevated, suggesting that caffeine may affect GABAergic synaptic transmission by modulating [Ca2+]i regulatory mechanisms. A representative Ca2+-dependent modulation of GABAergic transmission is the retrograde suppression of GABA release by endogenous cannabinoid (eCB). The working hypothesis of this project is that caffeine interacts with the brains'eCB system in the regulation of GABA-release by priming the Ca2+ source(s) necessary for the synthesis and release of eCB, thus negatively controlling the release of GABA. In order to test this hypothesis, three Specific Aims are proposed: 1) To isolate the signaling mechanisms of caffeine-induced Ca2+ release in the initiation of eCB synthesis, 2) To determine the role of caffeine on the induction of CRAC (calcium release-activated calcium entry) and its functional coupling to the synthesis of eCB, 3) To identify the effect of caffeine on the eCB-mediated synaptic plasticity and learning in the hippocampus. eCB is an emerging new candidate for the control of the brain's reward system and limbic emotional memory and learning. Caffeine has a prominent effect on the reward circuits sharing neurochemical properties with cannabinoid. This suggests there is a potential that caffeine interacts with the brain's endogenous cannabinoid system. Accomplishing the proposed Aims will increase our understanding of the ionic and molecular mechanisms underlying the physiological interaction between caffeine and the brain's endogenous cannabinoid system, and fill the gaps in the current knowledge on the regulation of GABAertic transmission. This project is dedicated to new discoveries on the physiological roles of two psychoactive compounds (caffeine and eCB) in the synaptic function and plasticity of central neurons in health and diseases including substance abuse, maladaptive learning, and addiction.